Genetic engineering strain producing lipase and construction method thereof

A technology of genetic engineering strain and construction method, which is applied in the field of lipase-producing genetic engineering strain and its construction, can solve the problems of high price and high price of biodiesel, and achieve the effect of reducing production cost

Inactive Publication Date: 2012-06-20
HEILONGJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims to solve the problem that most of the existing lipases are imported and the price is high, which leads to the high price of biodiesel, and provides lipase-producing genetic engineering strains and its construction method

Method used

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  • Genetic engineering strain producing lipase and construction method thereof
  • Genetic engineering strain producing lipase and construction method thereof
  • Genetic engineering strain producing lipase and construction method thereof

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Experimental program
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Effect test

specific Embodiment approach 1

[0011] Specific embodiment one: the cell of this embodiment produces lipase genetic engineering strain is ellipse, germinates and reproduces, forms ascospore; High lipase activity.

[0012] Pick a ring of lipase-producing genetically engineered strains in this embodiment, transfer them to the seed medium, place them on a shaker at 28°C and 180r / min for 24h, and obtain the seed solution; inoculate with 3% (v / v) Inoculate the seed solution into the fermentation medium, shake and culture at 28°C for 72 hours to obtain the fermentation liquid, centrifuge the fermentation liquid at 5000r / min and 4°C for 5min, and take the supernatant to obtain the crude enzyme liquid. The titration method was used to measure the crude enzyme liquid, and the lipase activity of the lipase-producing genetically engineered strain of this embodiment was 322.91-335.95 U / mL. The seed medium and fermentation medium are YPD medium.

specific Embodiment approach 2

[0013] Specific embodiment two: the construction method of this embodiment produces lipase genetic engineering strain, carries out according to the following steps: 1, the extraction of lipase producing bacterial strain Yarrowia lipolytica HDY-76 genomic DNA: get 3mL Yarrow lipolytica The bacterial liquid of Saccharomyces cerevisiae HDY-76 was centrifuged at 12000r / min for 1min, the supernatant was discarded, and the genomic DNA of Yarrowia lipolytica HDY-76 was extracted according to the genomic DNA extraction kit of TIANGEN company; 2. The lip7 gene PCR amplification: Use the genomic DNA extracted in step 1 as a template to perform PCR amplification to obtain amplified products, which are detected by 1% agarose gel electrophoresis; the amplified products are purified using a gel recovery kit , the target gene lip7 was obtained, and the target gene lip7 was sequenced to verify its accuracy; 3. The construction of the recombinant expression vector pGAPHαM-lip7: the target gene ...

specific Embodiment approach 3

[0028]Specific embodiment three: the difference between this embodiment and specific embodiment two is: the reaction system of PCR amplification in step 2 is as follows:

[0029]

[0030] PCR amplification conditions were: 94°C pre-denaturation for 4 min, 94°C denaturation for 45 s, 58.5°C annealing for 30 s, 72°C extension for 80 s, a total of 32 cycles, 72°C extension for 10 min, and 4°C incubation. Others are the same as in the second embodiment.

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Abstract

The invention relates to a genetic engineering strain producing lipase and a construction method thereof. The invention aims to solve the problem that as most of the existing lipase is imported and is expensive, biodiesel is more expensive. The cell of the genetic engineering strain producing lipase is elliptic and adopts budding reproduction to form ascospores; the spores are shaped like short mallets; the bacterial colony is circular, milky and non-transparent, and the surface is smooth and wet; and the enzyme activity of the produced lipase is high. The method comprises the following stepsof: extracting the DNAs (deoxyribonucleic acid) of the lipase-producing strain yarrowia lipolytica HDY-76 genome; performing polymerase chain reaction (PCR) amplification of the lip7; constructing the recombinant expression vector pGAPH alpha M-lip7; and constructing the genetic engineering strain producing lipase. The enzyme activity of the lipase produced by the constructed genetic engineering strain producing lipase is high. According to the invention, a foundation is laid for the application that the strain is used to catalyze oil through enzymolysis and produce biodiesel, thus the production cost of biodiesel can be reduced.

Description

technical field [0001] The invention relates to a lipase-producing genetic engineering strain and a construction method thereof. Background technique [0002] Lipase lipase is a special ester bond hydrolase, which can decompose various natural oils and fats produced by organisms, mainly hydrolyzing triglycerides formed by glycerol and insoluble long-chain fatty acids with more than twelve carbon atoms; at the same time , It can also catalyze the transesterification reaction of triglycerides and lower alcohols. Due to the catalytic properties of lipase, it can be used in the production of biodiesel. [0003] Biodiesel is a new type of renewable biomass energy that has been studied in recent years and has properties similar to diesel. Biodiesel mainly uses vegetable oil, animal oil and waste edible oil as raw materials. The production methods can be divided into direct mixed use, microemulsion method, pyrolysis method and transesterification method. Lipase can be used in ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/55C12N15/63C12R1/645
Inventor 赵凯
Owner HEILONGJIANG UNIV
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